Experimental design in the field of molecular diagnostics have evolved at an incredible rate over the last two decades. Analyses which historically took weeks or months to complete can now be completed within a day, resulting in a substantial savings of both time and costs. In addition to these spectacular advantages, the technology has progressed in another important parameter, which is one of scale. Through an improved understanding of electrochemistry, thermodynamics and physics, technicians have now achieved the capacity to isolate and observe particles at the nanoscale, and beyond.
Typically, a given biological sample must be thoroughly prepared prior to analysis, with such preparation being burdensome and, in a worst case scenario, unintentionally affecting the integrity of the sample to be analyzed. For example, many diagnostic assays on clinical samples containing biological components, such as blood, tissue or cells, require separation of the particles of interest from the crude sample by disrupting or lysing the cells to release such molecules including proteins and nucleic acids of interest, followed by purification of such proteins and/or nucleic acids. Only after the completion of such processing steps can analysis of the molecules of interest be initiated.
It is well known that certain forces may be applied to a sample in solution during processing in order to separate such sample into its component parts. One of the forces is known as dielectrophoresis (DEP) and is particularly useful once the experimental scale is performed in the micro- or nanoscale range, or if cells are utilized in the sample. DEP occurs when a polarizable particle is suspended or subjected to a non-uniform electric field (Kirby, B J, Micro- and Nanoscale Fluid Mechanics: Transport in Microfluidic Devices, Cambridge Univ Press (2010)). All particles exhibit some DEP activity in the presence of a non-uniform electric field, with the strength of such DEP force being highly correlated with several factors including, among others, the size, shape and electrical properties of the particles, frequency of the electric field and the solution in which the sample particles are being examined. Subjecting particles in solution to an electric field, where the electric field is set at a particular frequency, allows for specific and selective processing of the particles in solution, thereby allowing for very precise measurements to be obtained from the particles.
Prior art devices utilizing DEP forces to isolate and examine particles at the microscale are well known. Such devices include use of a glass slide having exposed electrodes plated on the slide surface, across which microliter quantities of fluid flows containing particles for analysis. These particles range from cells or proteins to nucleic acids, with such particles being capable of separation using DEP forces based on each particle's respective dielectric properties through use of separation buffers having specific conductivity and an external current (AC signal) having an appropriate amplitude and frequency.
These prior art devices, however, come with various problems, including binding of particles to exposed portions of the glass surface and, at times, the electrodes themselves. Additionally, the surfaces and electrodes of such prior art devices are quite small, resulting in the potential of aggregates of even the smallest of particles interfering with fluid flow and blocking certain processing steps between wash cycles.
The above mentioned prior art devices are based on microchip arrays attempting to take measurements of microliter quantities. There are macro-scale devices that have employed high voltage DC pulses in order to separate and analyze proteins and nucleic acids. While such devices generally overcome the blockage limitations of microscale arrays on a chip, the macroscale devices come with their own set of unique disadvantages. For example, some commercial macroscale devices use lysis conditions that limit the molecular weight of nucleic acids allowed for passage through pores created in the cell membranes. Additionally, released nucleic acids are often lost due to their non-specific binding to the surface of the lysis chamber. Furthermore, most macroscale devices require the use of a membrane or hydrogel which sits between the solution and electrodes, resulting in further limitations to such devices.
Most advanced devices in the prior art have attempted to take advantage of the various phenomena found within the field of electrokinetics relative to microfluidics. Electrokinetics describes the combination of DEP, electro thermal flow (ETF), electro osmotic flow (EOF) and other forces acting on particles found in a fluid as a function of frequency and amplitude of an applied electric field. One limitation found within even the most sophisticated devices in the prior art is the requirement that the DEP/ETF/EOF forces be coupled to one another. Other limitations include a restricted geometric configuration of electrode arrays based on a two-lead system, an inability to overcome the frequency and amplitude limitations found within high salinity systems (such as biological fluids) and poor processing of fluids as the salinity levels decrease to that of deionized water.
There is a frequency (less than 30 kHz) and amplitude (less than 20 Vpp) limit to capturing particles using electrokinetics in fluids with a conductivity greater than 1 mS/cm.
The frequency limit reduces the amount of mixing possible in the solution. As a result, there is an exponential decrease in the available concentration of particles present in the DEP depletion zone as a function of time. The DEP depletion zone is where particles can be influenced by the DEP force. This exponential decrease in concentration negatively influences capture efficiency of the devices exploiting electrokinetics for particle isolation, quantification, and recovery.
The amplitude limit reduces the potential DEP trapping force because at low frequencies electrode destruction is caused by electrolysis (strong changes in pH at the electrode locations) and destroys the electrodes, thereby removing their ability to function. The strong changes in pH can also cause changes to particles able to be captured by either altering their native state or destroying them. The amplitude limit is also required to balance the ability for the DEP forces to counter act the flow forces generated by ETF and EOF. As voltage (V) increases, DEP force increases as a function of V′ whereas flow forces increase as a function of V4 or V5 depending on fluid conductivity (Loire et al., A theoretical and experimental study of ac electrothermal flows, J. Phys. D: Appl. Phys., 45: 185301 (2012); Hong et al., Numerical simulation of AC electrothermal micropump using a fully coupled model, Microfluid Nanofluid, 13: 411-420 (2012)).
There is a limit to the amount of mixing that is possible in fluids with conductivity lower than 1 mS/cm generated by electro thermal flow due to a large reduction in Joule heating. This also limits the available concentration of particles in the DEP depletion zone. Increasing the amplitude of the electric field helps to increase the effective size of the DEP depletion zone, but this has a limit due to the generation of electrolysis.
There remains a need in the art for macroscale devices capable of sample processing down to the nanoscale level, while resolving the limitations described above.